During the past year we have addressed two critical questions: Does ORC recognize specific, genetically required, sequences in complex eukaryotic replication origins? Is ORC activity regulated as a function of the cell division cycle? The current paradigm for eukaryotic replication origins is the budding yeast (Saccharomyces cerevisiae). S. cerevisiae replication origins consist of ~0.1 kb regions that contain a genetically required consensus sequence to which ORC binds and an easily unwound DNA region where DNA synthesis begins. In contrast, replication origins in mammalian cells are much larger, and they lack a genetically required consensus sequence. Moreover, although they do contain genetically required sequences, and ORC is bound to specific sites in vivo, site specific ORC binding has not been observed in vitro. In an effort to understand the function of large complex eukaryotic replication origins, we turned our attention to fission yeast (Schizosaccharomyces pombe). S. pombe, like mammalian cells, contain large, AT-rich replication origins that lack a recognizable consensus sequence, but that nevertheless contain sequences that are required for replication. Others had shown that the N-terminal half of SpOrc4 contains nine AT-hook motifs that strongly bind to AT-rich DNA sequences. We found that SpORC binds to specific sites within S. pombe replication origins, both in vitro and in vivo, that are genetically required for origin activity, and that site selection is determined solely by the SpOrc4 subunit. These sites consisted of asymmetric A:T-rich sequences (clusters of A or T residues on one strand), but were devoid of either alternating A and T residues or GC-rich sequences. DNA binding specificity was independent of either ATP or the remaining five SpORC subunits. We further found that SpOrc4p binds specifically to only two of the four required sequences in the S. pombe replication origin ARS3001. A pre-RC is assembled adjacent to the strongest ORC binding site and this becomes the site where bi-directional DNA replication begins. The second, and weaker, SpOrc4 DNA binding site may facilitate pre-RC assembly, and the fourth required sequence appears to bind a non-ORC protein that is required for origin activation. Finally, we were able to show that frog (Xenopus laevis) ORC preferentially binds to the same AT-rich sites in sperm chromatin during its replication in frog egg extract that are targeted by SpOrc4. These results extend the classic paradigm for budding yeast to that of fission yeast, and suggest that fission yeast replication origins provide a more appropriate paradigm for the complex replication origins in multicellular animals. Taken together with other results from this and other labs, site specific DNA replication in multicellular organisms appears to result from both genetic and epigenetic parameters. ORC preferentially binds to asymmetric A:T-rich sequences, but during animal development some of these sites are selectively activated while others are suppressed. Is ORC activity regulated? Binding of ORC to DNA (chromatin) is the first step in assembly of a pre-replication complex. In contrast to yeast where all six ORC subunits are stably bound to chromatin throughout the cell cycle, mammalian Orc1 is selectively released from chromatin during S-phase. Moreover, the Orc1 that is released during S-phase is rapidly ubiquitinated and in some cases degraded. Other ORC subunits remain stably bound to chromatin and are not substrates for ubiquitination. During the M to G1 transition, Orc1 rebinds tightly to hamster ORC/chromatin sites to allow assembly of pre-replication complexes. This sites are located at specific genomic loci referred to as ?origins of bi-directional replication?. The role of ubiquitination is to sequester Orc1 during S-phase, and thus prevent reinitiation at replication origins during a single cell division cycle. This also provides a mechanism for reprogramming replication origins during animal development or as a result of DNA damage in which the Orc1 subunit could be degraded. In searching for the trigger that releases Orc1 from mammalian chromatin, we discovered two parameters that regulate the affinity of ORC for DNA in a cell cycle dependent manner. First, the affinity of Xenopus ORC for DNA depends on chromatin structure. With sperm chromatin, XlORC remains bound when pre-RCs are assembled, but its affinity becomes salt sensitive and it is released only during mitosis. With somatic cell chromatin, XlORC is released as soon as the pre-RC is assembled. These results demonstrated clearly the existence of an ?ORC cycle? that can regulate initiation of DNA replication during the cell cycle. In mammalian cells, ORC activity is regulated during cell division by selectively releasing the Orc1 subunit during S-phase and then preventing it from rebinding stably to chromatin until cyclin B is degraded, mitosis is completed, and a nuclear membrane is assembled. We have found that Orc1 is selectively and stably bound to a protein kinase during the G2/M-phase of proliferating hamster cells. This Orc1-associated protein kinase was identified as Cdk1(Cdc2)/cyclin A by its cell cycle specificity, ATP-binding, size, antibody recognition, immuno-precipitation with either anti-Orc1 or anti-Cdk1 antibodies, and sensitivity to inhibitors. It phosphorylated Orc1 in vitro, thereby accounting for the hyperphosphorylated Orc1 in M-phase cells that was dephosphorylated during the M to G1 transition. Orc1 in M-phase cells did not associate with either Orc2 or chromatin, but inhibition of Cdk activity resulted in rapid binding of Orc1 to chromatin. Thus, in mammals, premature rebinding of Orc1 to ORC/chromatin sites during mitosis appears to be prevented by the activity of Cdk1/cyclin A.